Oral delivery of bacteria expressing wsv108 gene-specific dsRNA protects shrimp from white spot syndrome virus (WSSV) infection.

Double-stranded RNA (dsRNA) can specifically inhibit gene expression by RNA interference and has important application potential in animal disease control. White spot syndrome virus (WSSV) is one of the most harmful pathogens in shrimp aquaculture, causing huge economic losses every year. In this study, we investigated the function of the WSSV-encoded wsv108 protein. We demonstrated that wsv108 could promote apoptosis by interacting with heat shock protein 70 (HSP70) and enhancing the expression of multiple apoptosis-related genes. Silencing of wsv108 gene by injection with specific dsRNA prepared by in vitro transcription significantly increased the survival rate of WSSV-infected shrimp and reduced the viral load in tissues, suggesting that wsv108 is important for WSSV pathogenicity. Based on this, we expressed the wsv108 specific dsRNA in engineered Escherichia coli. Oral feeding of this bacterium could inhibit the expression of wsv108, increase the survival rate of WSSV-infected shrimp, and decrease the viral load of WSSV in tissues. Therefore, this study developed a new method for treatment of WSSV disease by oral administration of bacterially expressed dsRNA against a novel therapeutic target molecule, which could be a potential candidate strategy for WSSV control in aquaculture.

Multi-function screening of probiotics to improve oral health and evaluating their efficacy in a rat periodontitis model.

The oral cavity is the second most microbially rich region of the human body, and many studies have shown that there is a strong association between microorganisms and oral health. Some pathogenic bacteria produce biofilms and harmful metabolites in the mouth that may cause oral problems such as oral malodor, periodontitis, and dental caries. Altering the oral microbiota by using probiotics may alleviate oral health problems. Thus, using multi-function screening, we aimed to identify probiotics that can significantly improve oral health. The main parameters were the inhibition of pathogenic bacteria growth, inhibition of biofilm formation, reduction in the production of indole, H2S, and NH3 metabolites that cause halitosis, increase in the production of H2O2 to combat harmful bacteria, and co-aggregation with pathogens to prevent their adhesion and colonization in the oral cavity. Tolerance to cholic acid and choline was also assessed. Bifidobacterium animalis ZK-77, Lactobacillus salivarius ZK-88, and Streptococcus salivarius ZK-102 had antibacterial activity and inhibited biofilm production to prevent caries. They also improved the oral malodor parameter, H2S, NH3, and indole production. The selected probiotics (especially L. salivarius ZK-88) alleviated the inflammation in the oral cavity of rats with periodontitis. The analysis of the gingival crevicular fluid microbiome after probiotic intervention showed that B. animalis ZK-77 likely helped to restore the oral microbiota and maintain the oral microecology. Next, we determined the best prebiotics for each candidate probiotic in order to obtain a formulation with improved effects. We then verified that a probiotics/prebiotic combination (B. animalis ZK-77, L. salivarius ZK-88, and fructooligosaccharides) significantly improved halitosis and teeth color in cats. Using whole-genome sequencing and acute toxicity mouse experiments involving the two probiotics, we found that neither probiotic had virulence genes and they had no significant effects on the growth or development of mice, indicating their safety. Taking the results together, B. animalis ZK-77 and L. salivarius ZK-88 can improve oral health, as verified by in vivo and in vitro experiments. This study provides a reference for clinical research and also provides new evidence for the oral health benefits of probiotics.

The Effects of Perioperative Probiotics on Postoperative Gastrointestinal Function in Patients with Brain Tumors: A Randomized, Placebo-Controlled Study.

The incidence of postoperative gastrointestinal dysfunction among neurosurgical patients is as high as 80%. Probiotics help to maintain gastrointestinal barrier defense, provide competitive adherence to mucus and epithelial cells, and regulate gastrointestinal motility. Therefore, the purpose of this study was to investigate whether probiotics enhance gastrointestinal health after craniotomy in patients with brain tumors. This study was a 15-day, prospective, randomized, double-blind, placebo-controlled trial for patients being treated with elective craniotomy for brain tumors. Participants were randomly divided into the probiotics group (4 g probiotics, twice daily) and placebo group. The primary outcome was the time of first stool after surgery. The secondary outcomes included assessments of the gastrointestinal function, changes in gastrointestinal permeability and clinical outcomes. We enrolled a total of 200 participants (probiotics: 100; placebo: 100) and followed the principles of intention-to-treat analysis. The time of first stool and flatus were significantly shorter in the probiotics group compared to the placebo group (P < 0.001, respectively). No significant trends were observed for any other of the secondary outcome variables. Our findings suggest that probiotics can improve the gastrointestinal mobility of patients received craniotomy, and this improvement cannot be explained by changes in gastrointestinal permeability.

Red mud recycling by Fe and Al recovery through the hydrometallurgy method: a collaborative strategy for aluminum and iron industry.

In this work, a collaborative strategy for the aluminum and iron industry based on red mud recycling through the hydrometallurgy method was proposed. In this method, Fe3+ and Al3+ were firstly separated from the red mud by using H2SO4 as a leaching agent, which was by-produced from the sintering process of an iron and steel industry. Multiple influence factors on the leaching process were investigated, with the H2SO4 addition amount showing the strongest influence on the leaching rates of Al and Fe. The main components of the filter residue were CaSO4, TiO2, and SiO2, which could be reused as additives in the building materials. Subsequently, the final Fe recovery product was obtained through the co-precipitation, Fe/Al separation, and Fe(OH)3 calcination. In the final product, the content of Fe2O3 reached 82.87%, and the iron grade was 58.01%, meeting the requirement being raw materials for sinter production.

White Spot Syndrome Virus (WSSV) Inhibits Hippo Signaling and Activates Yki To Promote Its Infection in Penaeus vannamei.

White spot syndrome virus (WSSV) is a serious threat to shrimp aquaculture, especially Pacific white shrimp, Penaeus vannamei, the most farmed shrimp in the world. Activation of the Hippo-Yki signaling pathway, characterized by the intracellular Hippo-Wts kinase cascade reactions and the phosphorylation and cytoplasmic retention of Yki, is widely involved in various life activities. The current work established the fundamental structure and signal transduction profile of the Hippo-Yki pathway in P. vannamei and further investigated its role in viral infection. We demonstrated that WSSV promoted the dephosphorylation and nuclear translocation of Yki, suggesting that Hippo signaling is impaired and Yki is activated after WSSV infection in shrimp. In vivo, Yki gene silencing suppressed WSSV infection, while Hippo and Wts silencing promoted it, indicating a positive role of Hippo signaling in antiviral response. Further analyses showed that Yki suppressed Dorsal pathway activation and inhibited hemocyte apoptosis in WSSV-infected shrimp, while Hippo and Wts showed opposite effects, which contributed to the role of Hippo signaling in WSSV infection. Therefore, the current study suggests that WSSV annexes Yki to favor its infection in shrimp by inhibiting Hippo signaling. IMPORTANCE White spot syndrome virus (WSSV) is one of the most harmful viral pathogens to shrimp. The pathological mechanism of WSSV infection remains unclear to date. The Hippo-Yki signaling pathway is important for various biological processes and is extensively involved in mammalian immunity, but little is known about its role in infectious diseases in invertebrates. Based on revealing the fundamental structure of the shrimp Hippo pathway, this study investigated its implication in the pathogenesis of WSSV disease. We demonstrated that WSSV enhanced Yki activation by inhibiting Hippo signaling in shrimp. The activated Yki promoted WSSV infection by inhibiting hemocyte apoptosis and suppressing the activation of Dorsal, an NF-κB family member in shrimp that is critical for regulating antiviral response. Therefore, this study suggests that WSSV can hijack the Hippo-Yki signaling pathway to favor its infection in shrimp.

A novel chitinase Chi6 with immunosuppressive activity promotes white spot syndrome virus (WSSV) infection in Penaeus vannamei.

Chitinases, a group of glycosylase hydrolases that can hydrolyze chitin, are involved in immune regulation in animals. White spot syndrome virus (WSSV) causes huge losses to crustacean aquaculture every year. We identified a novel chitinase Chi6 from Pacific white shrimp Penaeus vannamei, which contains a catalytic domain but no chitin-binding domain. The Chi6 expression was regulated by multiple immune signaling pathways and increased after immune stimulations. Silencing of Chi6 by RNAi in vivo did not affect Vibrio parahaemolyticus infection, but significantly increased the survival rate of WSSV-infected shrimp. The expression of multiple WSSV immediate early and structural genes was also decreased upon Chi6 silencing. The recombinant Chi6 protein showed no effect on bacterial growth but could attenuate shrimp hemocyte phagocytosis. The mRNA levels of several key elements and downstream genes of the MAPK and Dorsal pathways in Chi6-silenced shrimp were significantly up-regulated, suggesting an inhibitory effect of Chi6 on humoral immune response. Moreover, Chi6 enhanced the regulatory effect of Dorsal on the expression of WSSV ie1 gene. Therefore, Chi6 promotes WSSV infection through immunosuppression and regulation of WSSV gene expression. Targeting Chi6 could be a potential strategy for controlling WSSV disease in shrimp farming.

The Hippo-Yki Signaling Pathway Positively Regulates Immune Response against Vibrio Infection in Shrimp.

In the Hippo pathway, activation of Hippo and Warts (Wts) kinases results in the phosphorylation of Yorkie (Yki), to prevent its nuclear translocation. Shrimp aquaculture is threatened by Vibrio genus bacteria. In this study, we examine the role of the Hippo pathway in immune defense against Vibrio parahaemolyticus in Pacific white shrimp Penaeus vannamei. We show that V. parahaemolyticus infection promotes the expression of Yki and facilitates the dephosphorylation and nuclear translocation of Yki, indicating the inhibition of Hippo signaling upon bacterial infection. There is a complex regulatory relationship between the Hippo pathway components Hippo, Wts, and Yki and the immune-related transcription factors Dorsal, Relish, and STAT. Silencing of Hippo and Wts weakened hemocyte phagocytosis, while the silencing of Yki enhanced it, suggesting a positive regulation of shrimp cellular immunity by Hippo signaling activation. In vivo silencing of Hippo and Wts decreased the survival rates of V. parahaemolyticus-infected shrimp and elevated the bacterial content in tissues, while the silencing of Yki showed the opposite results. This suggests that the activation of Hippo signaling and the inhibition of Yki enhance antibacterial immunity in shrimp.

Sodium ascorbate as additive in red mud slurry for simultaneous desulfurization and denitrification: Insights into the multiple influence factors and reaction mechanism.

Based on the ultra-low emission demand of SO2 and NOx in flue gas, a new absorption method was proposed to improve the desulfurization and denitrification efficiency and reduce the amount of ozone by using sodium ascorbate as an additive in red mud slurry. Compared with pure red mud slurry, the red mud (RM) + sodium ascorbate (SA) slurry significantly improved the denitrification efficiency from 24% to 84% and the desulfurization efficiency to 98%. Meanwhile, the effects of RM, SA concentration, reaction time and O3/NO molar ratio on desulfurization and denitrification efficiencies were studied. The results showed that the RM + SA composite slurry maintained high efficiencies of desulfurization and denitrification for 240 min under the optimized conditions. As an antioxidant, the introduction of SA inhibited the excessive oxidation of sulfite, and itself could easily react with NO2 through the redox reaction, greatly promoting the absorption of NO2. In addition, the reaction mechanism of the simultaneous removal of SO2 and NO2 by red mud and sodium ascorbic mixed slurry combined was proposed.

A kelch motif-containing protein KLHDC2 regulates immune responses against Vibrio parahaemolyticus and white spot syndrome virus in Penaeus vannamei.

The kelch motif-containing proteins are widely present in organisms and known to be involved in various biological processes, but their roles in immunity remain unclear. In this study, a kelch motif-containing protein KLHDC2 was identified from Pacific white shrimp Penaeus vannamei and its immune function was investigated. The klhdc2 gene was widely expressed in shrimp tissues and its protein product was mainly present in the nucleus. Expression of klhdc2 was regulated by shrimp NF-κB family members Dorsal and Relish, and changed after immune stimulation. KLHDC2 could enhance the immune defense against Vibrio parahaemolyticus in shrimp but inhibit that against white spot syndrome virus (WSSV). Further analyses showed that KLHDC2 did not affect the phagocytosis of hemocytes but regulated the expression of a series of immune effector genes. KLHDC2 has a complex regulatory relationship with Dorsal and Relish, which may partly contribute to its positive role in antibacterial response by regulating humoral immunity. Moreover, the regulatory effect of KLHDC2 on WSSV ie1 gene contributed to its negative effect on antiviral response. Therefore, the current study enrichs the knowledge on the Kelch family and helps to learn more about the regulatory mechanism of shrimp immunity.

Simultaneous Removal of SO2 and NO x Using Steel Slag Slurry Combined with Ozone Oxidation.

In this work, steel slag slurry was used in combination with O3 oxidation for the simultaneous removal of SO2 and NO x in a laboratory-scale wet flue gas desulfurization process. The effects of the oxidation temperature, steel slag concentration, initial SO2 concentration, and pH value on the desulfurization and denitrification efficiencies were studied. The results showed that the highest NO x removal efficiency occurred at an oxidation temperature of 90 °C. With an increase of the oxidation temperature above 90 °C, the denitrification efficiency decreased due to the decomposition of N2O5. The effect of the SO2 concentration on denitrification was complicated. When the concentration of SO2 was 500 ppm, generation of SO3 2- promoted the absorption of NO2. However, higher SO2 concentrations strengthened the competitive absorption of SO2 and NO x . In the pH range of 8.5-4.5, the denitrification efficiency was maintained at about 96%. The component analyses of the aqueous solution and the solid residue were conducted to investigate the compositions of the absorption products. The results showed that NO3 - and SO4 2- were the major anions in the aqueous solution. The nitrogen balance was analyzed to be 95.8%, clearly illustrating the migration and transformation path of nitrogen. In the solid residue, most alkaline substances were consumed, and the final products were mainly CaSO4 and FeO. Accordingly, the reaction mechanism of simultaneous desulfurization and denitrification using steel slag combined with ozone oxidation was proposed.

The LARK protein is involved in antiviral and antibacterial responses in shrimp by regulating humoral immunity.

The LARK proteins containing a C2HC-type zinc finger motif and two RNA recognition motifs are conserved across vertebrates and invertebrates. Previous studies have suggested that invertebrate LARKs and their mammalian counterparts, the RBM4 proteins, regulate gene expression by affecting RNA stability and post-transcriptional processing, participating in multiple life processes. In the current study, the LARK gene from Pacific white shrimp Litopenaeus vannamei was identified and functionally explored in the context of immunity. The LARK protein was mainly present in the nucleus of its expression vector-transfected S2 cells, and the LARK mRNA was detectable in all the tested shrimp tissues. Expression of LARK in gill was up-regulated by immune stimulation with various pathogens. In vivo experiments demonstrated that LARK played positive roles in both antiviral and antibacterial responses and silencing of LARK could make shrimp more susceptible to infection with Vibrio parahaemolyticus and white spot syndrome virus (WSSV). Although silencing of LARK did not affect the phagocytic activity of hemocytes, it regulated expression of many components of the NF-κB and JAK-STAT pathways and a series of immune function proteins. These suggested that LARK could be mainly involved in regulation of humoral immunity. The current study could help reveal the roles of LARK/RBM4 in immunity and further explore the regulatory mechanisms of shrimp immunity.

Toll receptor 2 (Toll2) positively regulates antibacterial immunity but promotes white spot syndrome virus (WSSV) infection in shrimp.

The Toll family of receptors are a group of conserved pattern recognition receptors (PRRs) essentially controlling the initiation of innate immune responses. The white spot syndrome virus (WSSV) and Vibrio parahaemolyticus are major pathogens of aquaculture shrimp. Previous study has suggested that expression of the Toll2 receptor in Pacific white shrimp Penaeus vannamei was up-regulated by white spot syndrome virus (WSSV) infection but did not significantly changed upon infection with the bacterial pathogen Vibrio parahaemolyticus. The current study intends to investigate the role of P. vannamei Toll2 in antibacterial and antiviral immunity. We demonstrated that compared with the control, the Toll2-silenced shrimp was more susceptible to V. parahaemolyticus infection, suggesting that Toll2 may play a positive role in antibacterial immunity. However, silencing of Toll2 significantly enhanced survivorship of shrimp infected with WSSV and reduced the viral load in shrimp tissues. The expression of WSSV structural protein VP28 was also inhibited in Toll2-silenced shrimp. Histologic pathology analysis further showed that the WSSV infection was attenuated in stomach tissues from Toll2-silenced shrimp. These suggested that Toll2 could promote WSSV infection in shrimp. In Toll2-silenced shrimp, expression of antimicrobial peptides ALFs and PENs was significantly changed, which may contribute to the role of Toll2 in antibacterial immunity and WSSV infection.

A Novel Forkhead Box Protein P (FoxP) From Litopenaeus vannamei Plays a Positive Role in Immune Response.

The forkhead box protein P (FoxP) family members have been known to be important for regulation of immune responses in vertebrates, but their roles in invertebrate immunity remain unclear. In this study, a novel FoxP gene (LvFoxP) was identified from Pacific white shrimp Litopenaeus vannamei and functionally studied in the context of immune response. Possessing a conserved FoxP coiled-coil domain and a forkhead domain, LvFoxP shared homology to vertebrate FoxP family members, in particular FoxP1. Expression of LvFoxP was detectable in all the examined tissues and could be up-regulated by immune challenge in gill and hemocytes. The LvFoxP protein was present in both the cytoplasm and nucleus of hemocytes and could be nuclear-translocated upon immune stimulation. Silencing of LvFoxP increased the susceptibility of shrimp to infections by Vibrio parahaemolyticus and white spot syndrome virus (WSSV) and down-regulated the expression of multiple components of NF-κB and JAK-STAT pathways and almost all the examined immune effector genes. Moreover, the phagocytic activity of hemocytes from LvFoxP-silenced shrimp against V. parahaemolyticus was decreased. These suggested that LvFoxP could play a positive role in immune response. The current study may provide novel insights into the immunity of invertebrates and the functional evolution of the FoxP family.

A double chitin catalytic domain-containing chitinase targeted by c-Jun is involved in immune responses in shrimp.

Chitinases are a group of chitin-degrading enzymes widely distributed in organisms. Chitinases containing two chitin catalytic domains have been widely found in arthropods but their functions remain unclear. In this study, a member of these chitinases from Litopenaeus vannamei (dChi) was identified and functionally studied in the context of immunity. The promoter of dChi contained activator protein 1 (AP-1) binding sites and could be regulated by c-Jun. The recombinant dChi protein showed no bacteriostatic activity in vitro but knockdown of dChi in vivo increased the mortality of shrimp and the bacterial load in tissues after Vibrio parahaemolyticus infection, suggesting that dChi could play a positive role in antibacterial responses. However, silencing of dChi expression significantly decreased the mortality of WSSV-infected shrimp and down-regulated the viral load in tissues, indicating that dChi could facilitate WSSV infection. We further demonstrated that dChi was involved in regulation of the bacterial phagocytosis of hemocytes and expression of a series of immune related transcription factors and antimicrobial peptides. These indicated that the roles of dChi in antibacterial responses and anti-WSSV responses in vivo could result from its regulatory effects on the immune system. Taken together, the current study suggests that double chitin catalytic domain-containing chitinases could be important players in immune regulation in crustaceans.

Feasibility of anaerobic digestion for contaminated rice straw inoculated with waste activated sludge.

Anaerobic digestion of the heavy metal-contaminated rice straw inoculated with waste activated sludge was carried out through an orthogonal array. The results indicated that the digestion process was optimized with an inoculum to substrate ratio (I/S) of 0.25, a total solid (TS) content of 5%, and an initial pH of 7.0, with corresponding total biogas and methane yields of 745.4 and 285.2 mL/g VS, respectively. Under the optimized conditions, the bioleaching percentages of Cd and As in the biomass were 13.13% and 42.90%, respectively. According to range analysis, the biogas yield, methane yield, and bioleaching percentages of Cd and As in the biomass significantly decreased with an increase in TS content from 5% to 15%. The results suggested that anaerobic digestion with waste activated sludge was feasible for achieving both biogas production and bioleaching of heavy metals from contaminated rice straw.

A fiber-modified adenovirus co-expressing HSV-TK and Coli.NTR enhances antitumor activities in breast cancer cells.

Breast cancers especially in late and metastatic stages remain refractory to treatment despite advances in surgical techniques and chemotherapy. Suicide gene therapy based on adenoviral technology will be promising strategies for such advanced diseases. We previously showed that co-expression of herpes simplex virus thymidine kinase (HSV-TK) and Escherichia coli nitroreductase (Coli.NTR) by an hTERT-driven adenovirus vector resulted in additive anti-tumor effects in breast cancer cells in vitro and in vivo. As many tumor tissue and cancer cells express low level of coxsackie-adenovirus receptor (CAR), which is the functional receptor for the fiber protein of human adenovirus serotype 5 (Ad5), novel Ad5 vectors containing genetically modifi ed fiber are attractive vehicles for achieving targeted gene transfer and improving suicide gene expression in these cancer cells. In the present study, we first built a simplified Ad5 vector platform for fiber modification and quick detection for gene transfer. Then a fiber-modified adenovirus vector containing an RGD motif in the HI loop of the fiber knob was constructed. After recombined with HSV-TK and Coli.NTR gene, this fiber-modified Ad5 vector (Ad-RGD-hT-TK/NTR) was compared with that of our previously constructed Ad5 vector (Ad-hT-TK/NTR) for its therapeutic effects in human breast cancer cell lines. The anti-tumor activity of Ad-RGD-hT-TK/NTR was significantly enhanced compared with Ad-hT-TK/NTR both in vitro and in vivo. This new vector platform provided a robust and simplified approach for capsid modification, and the fiber-modified Ad5 with double suicide genes under the control of hTERT promoter would be a useful gene therapy strategy for breast cancer.